Complex polyketides such as 6-deoxyerythronolide B (6-dEB), the macrocyclic core of the antibiotic erythromycin, constitute an important class of natural products. They are synthesized by “modular” polyketide synthases, generally found in actinomycetes. For example, the polyketide synthase (PKS) which results in the synthesis of 6-dEB is produced in Sacromyces erythraea. The polyketides produced in these native hosts are generally subsequently tailored to obtain the finished antibiotic by glycosylation, oxidation, hydroxylation and other modifying reactions. Recent work from this laboratory has demonstrated that it is possible to express polyketide synthase modules in a functional form in Escherichia coli (Gokhale, R. S., et al., Science (1999) 284:482–485). However, in order to harness these modular enzymes for polyketide biosynthesis in E. coli, or in other hosts that do not normally produce them it is also necessary to produce their appropriate substrates in vivo in a controlled manner. For example, metabolites such as acetyl-CoA, propionyl-CoA, malonyl-CoA and methylmalonyl-CoA are the most common substrates of these enzymes. E. coli has the capability to produce acetyl-CoA, propionyl-CoA, and malonyl-CoA; however, the latter two substrates are only present in small quantities in the cell, and their biosynthesis is tightly controlled. The ability of E. coli to synthesize methylmalonyl-CoA has not been documented thus far.
Similar conditions prevail in other microbial cells, especially those that do not natively produce polyketides, such as various species of Escherichia, Bacillus, Pseudomonas, and Flavobacterium. Thus, generally, the required starter and/or extender units may not be produced in adequate amounts in any particular host. Further, by appropriate selection of the acyl transferase (AT) domains of the PKS in question, substrates more complex than those just mentioned may be employed. As an example, the PKS for synthesis of FK506 comprises an acyl transferase domain that incorporates substrates such as propyl malonyl-CoA in preference to malonyl-CoA or methylmalonyl-CoA. It would be helpful to have available a method which provides this range of substrates in appropriate levels in any arbitrarily chosen host organism.
Additional problems that may need to be surmounted in effecting the production of polyketides in procaryotic hosts, especially those which do not natively produce polyketides, include the presence of enzymes which catabolize the required starter and/or extender units, such as the enzymes encoded by the prp operon of E. coli, which are responsible for catabolism of exogenous propionate as a carbon and energy source in this organism. In order to optimize production of a polyketide which utilizes propionyl CoA as a starter unit and/or utilizes its carboxylation product, methylmalonyl CoA as an extender unit, this operon should be disabled, except for that portion (the E locus) which encodes a propionyl CoA synthetase. Any additional loci which encode catabolizing enzymes for starter or extender units are also advantageously disabled.
In addition, a particular procaryotic host, such as E. coli, may lack the phosphopantetheinyl transferase required for activation of the polyketide synthase. It may be required to modify the host to contain such a transferase as well.
In summary, it would be advantageous to effect the production of polyketides in microbial, especially procaryotic hosts in general, and, in particular, in hosts which do not natively produce polyketides. These hosts often have advantages over native polyketide producers such as Streptomyces in terms of ease of transformation, ability to grow rapidly in culture, and the like. These advantages are particularly useful in assessing the results of random mutagenesis or gene shuffling of polyketide synthases. Thus, the invention provides a multiplicity of approaches to adapt microbial hosts for the production of polyketides.